The present invention pertains to a method and apparatus for reliably, simply, rapidly and continuously measuring microbial activity in a microorganism-containing liquid.
The microorganism-containing liquid used in the present invention (hereinafter referred to as a culture liquor) means a culture liquor containing microorganisms or a microorganism suspension, prepared by separating living microorganism cells from a culture liquor and suspending the cells in another medium.
More particularly, the present invention relates to a method and apparatus for measuring microorganism activity, wherein the electrical properties originating from microorganism itself are measured by an electrochemical device.
Recently, fermentation processes are more widely being employed for the production of raw materials for food, medicine and the like. When such various useful compounds are produced by fermentation, it is very important in the effective and continuous performance of the fermentation process to measure the number or activity of microorganism cells in the culture liquor in each production step.
Heretofore, the conventional methods for measuring the number or activity of microorganisms in a fermentation batch were:
(1) Colony count method wherein the number of microorganism cells is determined by diluting the culture liquor, spraying the liquor on an agar plate medium and visually counting the number of colonies formed after a suitable incubation period. According to this method, it is possible to measure the number of microorganism cells, above all, the number of living cells.
(2) Optically measuring the turbidity of culture liquor caused by a Tyndall phenomenon and estimating the number of microorganism cells from degree of turbidity. According to this method, the number of the existing microorganism cells can be measured.
(3) Measuring the amount of adenosine triphosphate (ATP) enzymatically and estimating the number of microorganism cells therefrom. ATP, a high energy compound produced by microorganism cells in the culture liquor, can be regarded as being dependent upon the number of microorganisms and thus can be used as an index. According to this method, the number of living microorganism cells can be measured.
(4) Measuring the charged metabolites in the fermentation medium by determination of electric impedance. According to this method, the number of living microorganism cells can be obtained.
However, the foregoing known methods suffer from various disadvantages. For example, the colony count method is rather complicated in performance and operation, and it takes 24-48 hours of incubation before a count can be made. Moreover, the value obtained is not always reliable.
The method of measuring turbidity is based on an optical measurement, and a sample must be optically clear and isotropic. However, in industrial practices, media containing solid matters and colored media are more frequently used. Thus, it is actually very difficult to optically make an exact measurement of the number of cells.
According to the method wherein ATP is measured, luciferase is caused to react with ATP, and the fluorescent material thus formed are measured to obtain an amount of the corresponding ATP. As ATP or fluorescent materials exist in the medium it is difficult to exactly measure the amount of ATP originating from the microorganisms. As a result a reliable count of the number of microorganisms is not possible.
The method based on electric impedance suffers from the disadvantage that the measurement cannot be made under some aerobic conditions. Moreover, when a sample solution contains an electroconductive material, no measurement can be made. Since industrial fermentation processes are usually carried out under aerobic conditions, this method has no practical use.
In the cultivation of Actinomycetes and the like, many spores branch from individual cells with time, and many active parts appear. Similarly, the viscosity of the solution increases considerably with time, that is, with increasing number of cells and increasing branched spores. In the cultivation of such Actinomycetes, it is very important in the cultivation control to ascertain the number (activity) of the active parts rather than the number of cells, and no useful means for measuring the active parts was heretofore known.
As described above, the number or activity values of microorganisms so far measured according to any of the known methods has disadvantages, and it is the object of the present invention to realize a reliable and simple method for measuring microorganism activity in an industrial fermentation process.